1. Field of the Invention
This invention relates to the radioassay for vitamin B-12. More particularly, the invention relates to a radioassay for vitamin B-12 using the principle of radioisotope dilution in which improved techniques for the separation of vitamin B-12 not bound by intrinsic factor (IF) are provided as well as novel formulation of reagent solutions and their order of addition to achieve a more rapid and simpler method than those heretofore used in analogous assay procedures.
2. Description of the Prior Art
Principles of the methods to which the present improvements are directed are taught in the following references:
Lau, K.-S. Gottlieb, C. W., Wasserman, L. R., and Herbert, V., Measurement of Serum Vitamin B-12 Level Using Radioisotope Dilution and Coated Charcoal. Blood 26, 202 (1965) PA1 Herbert, V., Gottlieb, C. W., and Lau, K.-S, Hemoglobin-coated Charcoal Assay for Serum Vitamin B-12. Blood 28, 130 (1966) PA1 U.s. pat. No. 3,442,819. PA1 Dialysis PA1 Barakat, R. M., and Ekins, R. P. (1963). An Isotopic Method for the Determination of Vitamin B-12 Levels in Blood, Blood 21, 70-79 PA1 Friedner, S., Josephson, B., and Levin, K. (1969) Vitamin B-12 Determination by Means of Radioisotope Dilution and Ultrafiltration. Clin. Chim. Acta 24, 171-179 PA1 Diethylaminoethyl (DEAE) Cellulose PA1 Frenkel, E. P., Keller, S., and McCall, M. S. (1966) Radioisotopic Assay of Serum Vitamin B-12 with the Use of DEAE Cellulose, J. Lab. Clin. Med. 68, 510-522 PA1 Tibbling, G. (1969). A Method for Determination of Vitamin B-12 in Serum by Radioassay; Clin. Chim. Acta 23, 209-218 PA1 Protein Coated Charcoals PA1 Lau, K. S., Gottlieb, C. W., Wasserman, L. R. and Herbert, V. (1965) Measurement of Vitamin B-12 Level Using Radioisotope Dilution and Charcoal, Blood 26, 202-214 PA1 Herbert, V., Gottlieb, C. W. and Lau, K. S. (1966) Hemoglobin-coated Charcoal Assay for Serum Vitamin B-12, Blood 28, 1930-132 PA1 Raven, J. L., Walker, P. L., and Barkham, P. (1966) Comparison of the Radioisotope Dilution-coated Charcoal Method and a Microbiological Method (L. leichmanni) for Measuring Vitamin B-12 in Serum. J. Clin. Path. 19 610-613 PA1 Herbert, V., U.S. Pat. 3,442,819 (1969) PA1 Precipitation of Bound Vitamin B-12 PA1 Rothenberg, S. P. (1963), Radioassay of Serum Vitamin B-12 by Quantitating the Competition Between Co.sup.57 B-12 and Unlabelled B-12 for the Binding Sites of Intrinsic Factor. J. Clin. Invest. 42, 1391-1398 PA1 Sephadex PA1 Wide, L. and Killander, A. (1971). A Radiosorbent Technique for the Assay of Serum Vitamin B-12. Scand. J. Clin. Lab. Invest. 27, 151-159 PA1 Solid Phase-bound Intrinsic Factor PA1 Cesca, M., and Lundkvist, U., Use of Solid Phase Intrinsic Factor for Radiosorbent Assay of Vitamin B-12. Clin. Chim. Acta 32 (1971) 339-354 PA1 Ion Exchange Resins PA1 Brombacher, P. J., Gijzen, A. H. J., and Soons, M. P. J., A Systematic Investigation on the Assay of Vitamin B-12 in Serum by Radioisotope Dilution, Clin. Chim. Acta 36 (1972) 493-498 PA1 Roos, P., in Symposium on "in vitro" Procedures with Radioisotopes in Clinical Medicine, Vienna 1969. IAEA-SM-124/27.
In particular, the principle of the present assay known as radioisotope dilution is described in Example IX of said U.S. Pat. No. 3,442,819. In this procedure the serum vitamin B-12 and radioactive vitamin B-12 compete for limited binding capacity of IF. The portion of the radioactive vitamin B-12 and serum vitamin B-12 not bound must be removed. A number of previously reported methods of removing or separating the bound from the free vitamin B-12 are reported in the following:
All of these prior separation techniques have certain disadvantages. Thus, the use of charcoal solutions has detrimental effects as reported in "The Separation of Free and Bound Vitamin B-12", British Journal of Haematology, J. F. Adams and Fiona C. McEwan, 1974, 26, 581. In addition, the main disadvantages of charcoal suspensions is non-homogeneity of dose and introduction of additional suspension-carrying volume of fluid into each tube. Column methods also require time consuming elution, and may generate larger quantities of radioactive waste. Gel exclusion columns require exactly reproducible configuration, with respect to geometry and flow rate. Solid phase systems such as immobilized IF (such as those commercially available from Pharmacia) require prolonged agitation (about three hours) of a solid/liquid system and subsequent washing of the solid support to remove occluded material during centrifugation.
Substantially all of the foregoing difficulties are avoided by the present process in which bentonite, preferably in the convenient form of a pre-measured tablet, is utilized to adsorb unbound radioactive and serum vitamin B-12 . This adsorbent is easily separated from the assay media by centrifugation and results in uniformly high recoveries.